Nucleic acid encoding receptor type protein kinase

ABSTRACT

To provide a nucleic acid encoding a receptor protein kinase, wherein the nucleic acid has tandem duplication in a nucleotide sequence of a juxtamembrane and is useful for diagnosis of leukemia; a polypeptide encoded by the nucleic acid; an antibody capable of specifically binding to a region encoded by the nucleic acid having tandem duplication occurring in a nucleotide sequence of a juxtamembrane; a nucleic acid capable of specifically binding to the nucleic acid having tandem duplication occurring in a nucleotide sequence of juxtamembrane; a method for detection of the nucleic acid encoding a receptor protein kinase; and a kit therefor. A nucleic acid encoding a receptor protein kinase, wherein the nucleic acid has tandem duplication in a nucleotide sequence of a juxtamembrane; a polypeptide encoded by the nucleic acid; an antibody capable of specifically binding to the portion of the polypeptide; a nucleic acid capable of specifically binding to the nucleic acid; a method for detection of the nucleic acid; and a kit for detection.

This application is a divisional of co-pending U.S. application Ser. No.11/892,241, filed on Aug. 21, 2007. U.S. application Ser. No. 11/892,241is a divisional of application Ser. No. 10/653,147, which was filed onSep. 3, 2003, now abandoned, which is a divisional of U.S. applicationSer. No. 09/942,711 filed on Aug. 31, 2001, now U.S. Pat. No. 6,846,630,which issued on Jan. 25, 2005, which is a divisional of U.S. applicationSer. No. 09/284,654, filed on Apr. 16, 1999, under 35 U.S.C. §371(c),now abandoned. U.S. application Ser. No. 09/284,654 is a national phaseof PCT International Application No. PCT/JP97/03667 filed on Oct. 13,1997. PCT International Application No. PCT/JP97/03667 claims thebenefit of priority of Japanese Application No. 8-297329, filed on Oct.18, 1996, under 35 U.S.C. §119. The contents of the above applicationsare each incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention concerns a nucleic acid encoding a receptorprotein kinase, which has tandem duplication in a nucleotide sequence ofa juxtamembrane, a polypeptide, a method for detection of the abovenucleic acid and a kit for detection.

BACKGROUND ART

Proliferation and differentiation of cells, and responses of cells tovarious stimuli are strictly regulated by various growth factors. Thesegrowth factors are known to act via receptors which are specific to theabove growth factors (Nicola, N. A., Annu. Rev. Biochem. 58, 45, 1989;Lowenberg, B., Blood 81, 281, 1991). Of those receptors, the receptorscontaining a tyrosine kinase domain are classified as receptor tyrosinekinases (RTKs).

RTKs comprise an entracellular region, a transmembrane region, as wellas an intracellular region containing a tyrosine kinase domain and ajuxtamembrane between the transmembrane region and the tyrosine kinasedomain, and further roughly classified into four types according tostructural characteristics and amino acid sequence homology.

Type I receptors have a monomeric structure, with two cysteine-richrepeat sequences in their extracellular region, and are exemplified bythe EGF receptor, HER2/neu and the like.

Type II receptors have a structure comprising two subunits each for αand β, which are bound via S—S bond, wherein the α chain is anextracellular region containing one cysteine-rich repeat sequence, andwherein the β chain has a transmembrane region, a juxtamembrane, and atyrosine kinase domain. Examples are an insulin receptor and an IGF-1receptor.

Type III receptors have a monomeric structure containing fiveimmunoglobulin-like cysteine-rich sequences in their extracellularregion and two tyrosine kinase domains interrupted by a kinase insert intheir intracellular region. Examples are PDGF receptor, fms (CSF-1receptor), kit (SLF receptor) and the like.

Type IV receptors resemble type III receptors but have threeimmunoglobulin-like repeat sequences, and are exemplified by FGFreceptor.

fms-Like tyrosine kinase 3 (hereinafter abbreviated as FLT3; Matthews,W., Cell 65, 1143, 1991; Rosnet, O., Genomics 9, 380, 1991), which isexpressed in leukemic cells etc., also referred to as fetal liver kinase2 (FLK2) or STK-1, is known to as type III receptors (Small, D., Proc.Natl. Acad. Sci. USA 91, 459, 1993; Lyman, S. D., Oncogene 8, 815, 1993;Rosnet, O., Blood 82, 1110, 1993; Agnes, F., Gene 145, 283, 1994).

In these receptor tyrosine kinases, aggregation, such as dimerization,takes place upon binding of a ligand, such as a growth factor, to theextracellular region, thereby resulting in the activation of kinase.Although in these tyrosine kinases, their ligands have been first foundand then their receptors in most cases, there are receptors of whichligands remain unknown.

Regarding FLT3, which has been remarked in proliferation mechanism ofhematopoietic stem cells and leukemia, after finding the FLT3, the FLT3ligand has been found (Lyman, S. D., Cell 75, 1157, 1993; Hannum, C.,Nature 368, 643, 1994). Since the FLT3 ligand is expressed in almost allleukemic cells, it is assumed that cells are proliferated a mechanism ofautocrine stimulation in leukemia (Meierhoff, G., Leukemia 9, 1366,1995). Also, FLT3 mRNA has bean reported to be expressed in lymphaticleukemic dells and myelocytic leukemic cells (Birg, Blood 80, 2584,1994; Da Silva, N., Leukemia 8, 885, 1994; Drasel, K., Leukemia 9, 1212,1995; Drexler, H. G., Leukemia 10, 588, 1996). However, there remainsunknown how the FLT3 mRNA expression is associated with the pathology oflymphatic leukemia and myelocytic leukemia.

A human FLT3 cDNA has been cloned, and a cDNA nucleotide sequence andthe amino acid sequence of the FLT3 protein have been determined [O.Rosnet at al., Blood, 82(4), 1110-1119 (1993)]. The present situation,however, is that the structure and function of FLT during thehematopoietic stem cell differentiation and the malignant alterations toleukemic cells have not been analyzed well.

DISCLOSURE OF INVENTION

Accordingly, a first object of the present invention is to provide anucleic acid encoding a receptor protein kinase, wherein the nucleicacid has tandem duplication in a nucleotide sequence of a juxtamembraneand is useful for diagnosis of leukemia, and to provide a nucleic acidencoding the above juxtamembrane. A second object of the presentinvention is to provide a polypeptide which is encoded by the abovenucleic acid. A third object of the present invention is to provide anantibody capable of specifically binding to a portion encoded by anucleic acid having tandem duplication occurring in a nucleotidesequence of a juxtamembrane. A fourth object of the present invention isto provide a nucleic acid capable of specifically binding to a nucleicacid having tandem duplication occurring in a nucleotide sequence of ajuxtamembrane. A fifth object of the present invention is to provide amethod for detection of the nucleic acid encoding a receptor proteinkinase and a kit therefor.

Conventionally, as to the FLT3, the same receptor protein kinase isexpressed, irrespective of kinds of cells and differentiation [O. Rosnetet al., Blood, 82(4), 1110-1119 (1993)]. As a result of the detailedinvestigation and intensive studies of the FLT3 expression in leukemiccells, however, the present inventors surprisingly have found a receptorprotein kinase gene having novel tandem duplication in a juxtamembrane,and found that the above tandem duplication is somatic, and that theexpression of FLT3 having the above tandem duplication is associatedwith leukemia malignancy and mal-consequence of prognosis, and thepresent invention has been completed thereby.

Accordingly, the gist of the present invention is as follows:

[1] a nucleic acid encoding a receptor protein kinase, wherein thenucleic acid has tandem duplication in a nucleotide sequence of ajuxtamembrane;[2] the nucleic acid according to item [1] above, wherein the receptorprotein kinase is a receptor tyrosine kinase;[3] the nucleic acid according to item [2] above, wherein the receptortyrosine kinase is FMS-like tyrosine kinase 3 (FLT3);[4] the nucleic acid according to any one of items [1] to [3] above,wherein the nucleic acid comprises a nucleotide sequence encoding anamino acid sequence as shown by any one of SEQ ID NOs: 1 to 5 inSequence Listing in a juxtamembrane[5] the nucleic acid according to any one of items [1] to [3] above,wherein the nucleic acid comprises a nucleotide sequence as shown by anyone of SEQ ID NOs: 6 to 15 in Sequence Listing in a juxtamembrane;[6] a nucleic acid encoding a tandem duplication mutant of FLT3 as shownby any one of SEQ ID NOs: 16 to 20 in Sequence Listing;[7] a nucleic acid comprising a nucleotide sequence encoding a tandemduplication mutant as shown by any one of SEQ ID NOs: 21 to 25 inSequence Listing, or a nucleic acid capable of hybridizing thereto understringent conditions, wherein the nucleic acid has tandem duplication ina nucleotide sequence encoding a juxtamembrane;[8] a nucleic acid having tandem duplication, wherein the nucleic acidencodes an amino acid sequence as shown by any one of SEQ ID NOs: 1 to 5in Sequence Listing;[9] a nucleic acid as shown by any one of SEQ ID NOs: 6 to 15 inSequence Listing, or a nucleic acid capable of hybridizing thereto understringent conditions, wherein the nucleic acid has tandem duplication;[10] a polypeptide encoded by the nucleic acid according to any one ofitems [1] to [9] above;[11] a polypeptide comprising an amino acid sequence as shown by any oneof SEQ ID NOs: 1 to 5, and 16 to 20 in Sequence Listing;[12] a polypeptide encoded by a nucleic acid having tandem duplicationin a nucleotide sequence of a juxtamembrane, wherein the polypeptideresults from at least one of deletion, substitution or addition of oneor more amino acid residues in an amino acid sequence as shown by anyone of SEQ ID NOs: 1 to 5, and 16 to 20;[13] an antibody capable of specifically binding to a region encoded bya nucleic acid having tandem duplication occurring in a nucleotidesequence of a juxtamembrane of a receptor protein kinase;[14] a nucleic acid capable of specifically binding to a nucleic acidhaving tandem duplication occurring in a nucleotide sequence of ajuxtamembrane of a receptor protein kinase;[15] a method for detection of a'nucleic acid encoding receptor proteinkinase and having tandem duplication occurring in a nucleotide sequenceof a juxtamembrane, comprising:step (a): preparing a human nucleic-acid sample;step (b): subjecting the nucleic acid sample obtained instep (a) to gene amplification reaction to provide a nucleic acidfragment obtained by amplifying a region having tandem duplication in ajuxtamembrane which can be found in a nucleic acid encoding a receptorprotein kinase; andstep (c): examining the presence of tandem duplication for the nucleicacid fragment of step (b);[16] the method for detection according to item [15] above,characterized in that the method is utilized in diagnosis of M2, M4, orM5 based on the FAB (French-American-British) classification of acutemyeloid leukemia;[17] a kit for detection of a nucleic acid encoding a receptor proteinkinase and having tandem duplication in the nucleotide sequence of ajuxtamembrane, characterized in that the kit comprises primers foramplifying a region having tandem duplication, wherein the region can befound in the receptor protein kinase gene;[18] the kit according to item [17] above, characterized in that the kitis utilized in diagnosis of M2, M4, or M5 based on the FAB(French-American-British) classification of acute myeloid leukemia; and[19] use of the nucleic acid according to any one of items [1] to [9]above for detection of a nucleic acid encoding a receptor protein kinaseand having tandem duplication in a nucleotide sequence of ajuxtamembrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing agarose gel electrophoresis of the case whereRT-PCR is carried out with RNA obtained from leukemic cells derived fromAML patients as a template. In the figure, lanes 1 to 5 respectivelyshow results for patients belonging to M1, M2, M3, M4 and M5 (M34patients) on FAB classification, and lanes 6 to 9 respectively showresults for M1, M2 (M155 patients), M3 and M4 (M162 patients).

FIG. 2 is a schematic view showing tandem duplication at exon 11 andexon 12 for M34, M155, M162, M810 and M839.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained below.

The nucleic acid encoding a receptor protein kinase of the presentinvention has tandem duplication in a region encoding a juxtamembrane.The nucleic acid of the present invention encoding a protein kinase canbe a nucleic acid encoding either tyrosine kinase or serine-threoninekinase. For diagnosis of leukemia, preferred are nucleic acids encodinga receptor protein kinase, and nucleic acids encoding FMS-like tyrosinekinase 3 (FLT3) are preferably used.

In the present invention, the juxtamembrane is present between thetransmembrane region and the kinase domain of the receptor proteinkinase, and the juxtamembrane constructs an intracellular membraneregion together with the kinase domain [O. Rosnet, et al., Blood, 82(4), 1110-1119 (1993)].

In the present invention, tandem duplication refers to a nucleotidesequence in which an entire portion or partial portion of a nucleic acidencoding a juxtamembrane is repeated one or more times in the sameorientation. The above repeat nucleotide sequences can be lined up onedirectly after another, or they can contain optional nucleotidesequences between each of the repeat nucleotide sequences. In addition,the number of duplicated base is not particularly limited. Furthermore,although mutations of deletion, substitution or addition of one or morebases can exist in a portion of a nucleotide sequence between thecorresponding tandem duplications. In the tandem duplication of thepresent invention, the tandem duplication may be detected as lengthmutation. For example, the tandem duplication is contained in cDNAhaving nucleotide sequences of SEQ ID NOs: 6 to 10, and in genomic DNAhaving nucleotide sequences of SEQ ID NOs: 11 to 15 as nucleic acidsencoding a juxtamembrane.

Nucleic acids (cDNA or genomic DNA) having tandem duplication which arenewly found in a nucleotide sequence of the juxtamembrane of FLT3 arenamed M34 (SEQ ID NOs: 6 and 11), M155 (SEQ ID. NOs: 7 and 12), M162(SEQ ID NOs: 8 and 13), M810 (SEQ ID NOs: 9 and 14) and M839 (SEQ IDNOs: 10 and 15), respectively, and their schematic view is shown in FIG.2. Incidentally, it is desired that the tandem duplication in thepresent invention takes place in-frame. Amino acid sequences encoded bythe above SEQ ID NOs: 6 to 10 are shown in SEQ ID NOs: 1 to 5.

The nucleic acid of the present invention concerns a nucleic acidencoding a receptor protein kinase, wherein the nucleic acid has thetandem duplication as described above in a nucleotide sequence of ajuxtamembrane, particularly a nucleic acid of FMS-like tyrosine kinase 3(FLT3) mutant, wherein the nucleic acid has the tandem duplication in anucleotide sequence of a juxtamembrane. The amino acid sequences of ajuxtamembrane having the tandem duplication are shown by e.g. SEQ IDNOs: 1 to 5 as mentioned above, and the nucleic acids of the presentinvention are those comprising nucleotide sequences encoding these aminoacid sequences. Concretely, for example, the present nucleic acids arethose comprising nucleotide sequences shown by SEQ ID NOs: 6 to 15. Moreparticularly, a nucleic acid of a tandem duplication mutant of FLT3comprising a nucleic acid of a juxtamembrane includes, for example,nucleic acids encoding tandem duplication mutants of FLT3 shown by SEQID NOs: 16 to 20, more concretely, nucleic acids comprising nucleotidesequences encoding tandem duplication mutants of FLT3 shown by SEQ IDNOs: 21 to 25. In addition, they may be a nucleic acid capable ofhybridizing to the above nucleic acid under stringent conditions, andhaving tandem duplication in a nucleotide sequence encoding ajuxtamembrane.

Furthermore, the nucleic acid of the present invention concerns anucleic acid encoding a juxtamembrane and having tandem duplication, thenucleic acid including, for example, a nucleic acid having tandemduplication, wherein the nucleic acid encodes amino acid sequences shownby SEQ ID NOs; 1 to 5, concretely, nucleic acids shown by SEQ ID. NOs: 6to 15, or a nucleic acid has tandem duplication capable of hybridizingto those nucleic acids under stringent conditions.

Here, hybridizing under stringent conditions refers to hybridizationwith the nucleic acids, wherein the hybridization comprises, forexample, incubating a nucleic acid-immobilized membrane with a probe at50° C. for 12 to 20 hours in 6×SSC, wherein 1×SSC indicates 0.15 ⁻MNaCl, 0.015 M sodium citrate, pH 7.0, containing 0.5% SDS, 0.1% bovineserum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1% Ficol 400, and0.01% denatured salmon sperm DNA, but not limited to the aboveconditions.

The nucleic acid of the present invention can be obtained by, e.g. thefollowing method.

First, cells in which length mutation takes place are detected bysynthesizing cDNA by a reverse transcriptase with RNA as a template, theRNA purified from various pathologic cells, particularly leukemia cells,thereafter carrying out DNA amplification reaction using primers whichare targeted to a region encoding a juxtamembrane of a desired receptorprotein kinase, and comparing the length of the amplified DNA fragmentsby means of an electrophoresis method. Further, it is possible toidentify whether or not a mutation is tandem duplication by determininga nucleotide sequence of the obtained amplified DNA fragment.

Next, cDNA encoding a receptor protein kinase, the cDNA having noveltandem duplication of the present invention can be obtained bysynthesizing cDNA by a reverse transcriptase with RNA obtained fromcells in which tandem duplication takes place, thereafter carrying outDNA amplification reaction using primers which can specifically amplifycDNA of a desired receptor protein kinase.

The nucleic acid of the present invention can be also obtained by usinggenomic DNA as a template, the genomic DNA purified from pathologiccells.

In the present invention, leukemia cells are selected as the pathologiccells, and FLT3 is preferably targeted as the receptor protein kinase.

An FLT3 gene comprises 21 exons, and alternatively, the juxtamembrane isencoded in 18 by at 3′-side of exon 10 and 117 by at 5′-side of exon 11[O. Rosnet, et al., Oncogene, 6, 1641-1650 (1991)]. Primers covering theregion of exon 11 and exon 12 can be selected as primers used in DNAamplification reaction. Examples of the nucleotide sequences are shownin SEQ ID NOs: 26 and 27. Incidentally, exon 12 and 16 by at 3′-side ofexon 11 encode a partial portion of the tyrosine kinase domain.

DNA amplified fragments as shown by SEQ ID NOs: 6 to 10 are obtainedwhen RNA is used as a template for DNA amplification reaction, and DNA,amplified fragments as shown by SEQ ID NOs: 11 to 15 are obtained whengenomic DNA is used as a template. As a result, it is confirmed thatthese resulting fragments have in-frame tandem duplication within exon11 or exons 11 to 12.

Alternatively, nucleotide sequences of cDNA encoding a whole length ofFLT3 and having the above in-frame tandem duplication are shown in SEQID NOs: 21 to 25.

The polypeptide of the present invention is a polypeptide encoded by theabove nucleic acids. Concretely, there can be exemplified a polypeptidecomprising amino acid sequences of SEQ. ID NOs: 1 to 5, and tandemduplication mutants of FLT3 as shown by SEQ ID NOs: 16 to 20.

The polypeptide of the present invention can be obtained by purifyingfrom cells expressing the polypeptide, and can be also obtained byemploying a conventional gene engineering procedures. A tandemduplication mutant of FLT3, for example, can be obtained by insertingthe above nucleic acids into a suitable expression vector, and thenexpressing the product in a suitable host. In addition, a polypeptidewith only a juxtamembrane of a receptor protein kinase of the presentinvention can be obtained by inserting a DNA fragment encoding ajuxtamembrane alone to the above expression vector.

Further, the polypeptide of the present invention can be expressed as afusion protein. For instance, in order to increase amounts of expressionof a desired protein, N-terminal peptide chain derived from otherprotein is added to N-terminus of the desired protein, or a suitablepeptide chain is added to N-terminus or C-terminus of the desiredprotein to express the resulting polypeptide, so that purification ofthe desired protein using a resin carrier having affinity to the peptidechain can be facilitated.

The present polypeptide also encompasses a polypeptide encoded by anucleic acid having tandem duplication in a nucleotide sequence of ajuxtamembrane, wherein the polypeptide results from at least one ofdeletion, substitution or addition of one or more amino acid residues inamino acid sequences of the present invention, e.g. SEQ ID NOs: 1 to 5,16 to 20 in Sequence Listing. In other words, there can be a case whereno mutations take place in amino acid sequences in the region encoded bytandemly duplicated nucleic acids, and deletion, substitution oraddition of amino acid residues takes place in other portions of aminoacid sequences; or a case where deletion, substitution or addition ofamino acid residues takes place in amino acid sequences of the regionencoded by tandemly duplicated nucleic acids. Introduction of deletion,substitution or addition of the amino acid residues can be easilycarried out by introducing mutation into the desired nucleic acidsequence by a method using restriction endonucleases, nucleases and thelike, or a method for performing site-directed mutagenesis [W. Ito, etal., Gene, 102, 67-70 (1991)] etc., thereby incorporating the mutatednucleic acid sequence into an expression vector to express the productin a suitable host cell.

In the present invention, the antibody refers to an antibody capable ofspecifically binding to a region encoded by a nucleic acid having tandemduplication occurring in a nucleotide sequence of a juxtamembrane of thereceptor protein kinase. In order to obtain the antibody, for example,the antibody is obtained as anti-serum by immunizing animals with apeptide having amino acid sequences of SEQ ID NOs; 1 to 5 together withadjuvant by conventional method. In addition, the antibody can beobtained as a monoclonal antibody by a method described in G. Galfare,et al., Nature, 266, 550-552 (1997).

In the present invention, the nucleic acid capable of specificallybinding to nucleic acids having tandem duplication occurring in anucleotide sequence of a juxtamembrane of the receptor protein kinase isnot to be particularly limited, and is exemplified by antisense DNA ofdouble stranded DNA having tandem duplication or RNA corresponding tothe antisense DNA.

The method for detection of a nucleic acid of the present inventioncomprises the following steps:

step (a): obtaining a human nucleic acid sample;step (b): subjecting the nucleic acid sample obtained in the above step(a) to gene amplification reaction to provide a nucleic acid fragmentobtained by amplifying a region having tandem duplication in ajuxtamembrane,wherein the region can be found in a nucleic acid encoding a receptorprotein kinase; andstep (c): examining the presence of tandem duplication for the nucleicacid fragment of the above step (b).

First, step (a) will be described. The human nucleic acid sample usablein the present invention is not to be particularly limited, as long asit is a nucleic acid encoding a receptor protein kinase, the nucleicacid having tandem duplication in a nucleotide sequence of ajuxtamembrane, such as genomic DNA, cDNA or mRNA. The human nucleic acidsample can be prepared by conventionally performed known method,including, for instance, a method described in Molecular Cloning: ALABORATORY MANUAL, 2nd eds. (T. Maniatis et al., Cold Spring HarborLaboratory Press, published in 1989).

Secondly, step (b) will be described. The nucleic acid sample andsuitable primers are used to amplify a nucleic acid encoding a regioncontaining mutation site which can be found in a juxtamembrane of thereceptor protein kinase of the interest to obtain a desired nucleic acidfragment. A method for performing DNA amplification reaction usable inthis step is not particularly limited, as long as it is a method capableof amplifying the above region, and there can be utilized nucleic acidamplification methods, such as a nucleic acid amplification methodutilizing RT-PCR method, PCR method, or RNA polymerases (Japanese PatentLaid-Open Nos. Hei 2-5864 and Hei 7-203999), or strand substitutionamplification method (Japanese Examined Patent Publication No, Hei7-114718, and Japanese Patent Laid-Open No. Hei 7-88242). Among them,the RT-PCR method or PCR method is preferably used.

The region to be amplified having tandem duplication in a juxtamembraneincludes, for example, in case of FLT3, a region containing a whole orpartial portion of the region from 18 by at 3′-side of exon 10 to 117 byat 5′-side of exon 11, without being particularly limited thereto aslong as the region contains an exon 11 site.

The primers used in RT-PCR method or PCR method are not particularlylimited as long as they are primers capable of amplifying a DNA fragmentcontaining the above mutation site. Concretely, there can be exemplifieda primer pair having nucleotide sequences as shown in SEQ ID NOs: 26 and27 in Sequence Listing. In addition, PCR conditions are not particularlylimited, and conventionally performed known conditions can be used onPCR reaction.

Thirdly, step (c) will be described. In this step, the presence oftandem duplication for the nucleic acid fragment obtained in step (b) isexamined. The method for detection of the presence of tandem duplicationis not particularly limited, and it is preferable that a method ofcomparing lengths of amplified DNA fragments by means of agarose gelelectrophoresis method is used.

In addition, a method for examining single strand conformationpolymorphism (SSCP) can be used as a method for detection of mutationwhich is usable in this step. The method comprises examining thedifferences of a higher-order structure as the differences of mobilityin electrophoresis, wherein the high-order structure is dependent on anucleotide sequence in which single-stranded DNA is formed byintramolecular interaction (Proc. Natl. Acad. Sci. USA, 86: 2766-2770,1989). The presence or absence of mutation can be detected by subjectingthe nucleic acid fragment obtained in step (b) to electrophoresis underconditions described in the above-mentioned publication, and comparingits mobility with that of a nucleic acid fragment derived from a normalreceptor protein kinase.

Other detection methods include a method in which the above step (c) isaltered to other method for detection of mutation. For the detection ofmutation, there can be used a known method for detection of mutation,such as hybridization method using a suitable DNA fragment containing amutation site as a probe, or DGGE method [Val C, Sheffield et al., Proc.Natl. Acad. Sci. USA 86, 232-236 (1989)]. In addition, a method fordetection of mutation using a MutS protein is known (Japanese PatentLaid-Open No. Hei 7-327698).

The mutation can be identified by sequencing the nucleotide sequence forDNA fragment in which a length mutation is confirmed by means of theabove-mentioned method. For sequencing the nucleotide sequence, aconventionally used method can be employed, including, for example, amethod comprising cloning an amplified DNA fragment into a suitablevector and determining the nucleotide sequence, or a method fordetermining the nucleotide sequence using an amplified fragment per seas a template.

As described above, the present invention provides a use of a nucleicacid of the present invention described above for detection of a nucleicacid encoding a receptor protein kinase, wherein the nucleic acid hastandem duplication in a nucleotide sequence of a juxtamembrane.

The method for detection of a nucleic acid of the present invention canbe utilized in diagnosis of M2, M4, and M5 based on the FAB(French-American-British) classification of acute myeloid leukemia(AML). Based on the FAB (French-American-British) classification,pathologic types of AML are classified into six classes as M1(myeloblastic, no maturation potential), M2 (myeloblastic, withmaturation potential), M3 (promyelocytic), M4 (myelomonocytic), M5(monocytic), and M6 (erythroleukemia) (Shin Rinsho Kensa Gishi Koza, 10,Ketsuekigaku, 75, Igaku-Shoin).

It is understood that patients harboring an FLT3 gene having the tandemduplication of the present invention belong to classes M2, M4, and M5above, and that the patients relapse into symptom to death even withtransient symptom remission, so that their prognosis leads tomal-consequence. Therefore, according to the detection method of thepresent invention, there can be provided a method for examination usefulto the pathologic judgment of AML.

Incidentally, of the above patients, the detection of mutation usinggenomic DNA from myelocyte of a patient obtained at the time of symptomremission is carried out, and as a result, tandem duplications in ajuxtamembrane are not found. This mutation is therefore assumed to be asomatic mutation.

Also, the nucleic acid of the present invention, which has tandemduplication, serves as a marker for myelodysplastic syndrome (MDS),which develops in the pre-stage of leukemia, AML with dysplasia, and thelike, as well as AML as classified based on the FAB classification. Thedetection method of the present invention therefore is a method forexamination which is useful for the pathologic judgment of thesediseases.

By utilizing the above detection method, there can be provided a kit fordetection of a nucleic acid of the present invention. Concretely, thereis a kit for detection of a nucleic acid by the above describeddetection method, the nucleic acid encoding a receptor protein kinaseand having tandem duplication in the nucleotide sequence of ajuxtamembrane, characterized in that the kit comprises primers foramplifying a region having tandem duplication, wherein the region can befound on the receptor protein kinase gene.

The diagnosis of the above AML etc. can be easily carried out by usingsuch a kit.

In the present invention, a polypeptide encoded by a nucleic acid havingtandem duplication as described above can be further detected by thesteps shown below:

step 1: obtaining a human protein sample; andstep 2: examining the presence of tandem duplication in the nucleotidesequence of a juxtamembrane of the protein sample obtained in the abovestep 1.

First, step 1 will be described. The human protein sample can beprepared by preparing a membrane protein from a cell which is assumed tohave the polypeptide of the present invention expressed therein (e.g.,leukemic cell, in case of FLT3).

Second, step 2 will be described. The method for detection of tandemduplication mutations is not particularly limited, and can be carriedout by using a labeled antibody capable of specifically binding to thejuxtamembrane encoded by a nucleic acid having a tandem duplicationmutation.

This step can, for example, be carried out by a method comprisingsubjecting the protein sample obtained in step 1 to SDS-PAGE to separateproteins, and subsequently detecting the desired protein byimmunoblotting method.

In another embodiment of the present invention, there can be provided amethod for regulating the proliferation, immune response and signalinformation transmission of leukemic cells, hematopoietic stem cells,etc. using the above nucleic acids or polypeptides, or nucleic acids orantibodies capable of specifically binding thereto.

Among them, a preferred embodiment includes an application toimmunotherapy for tumors. Conventionally, it has been known thattumor-specific peptides of proteins specifically expressed in tumorcells serve as targets of T cell immune responses to tumor cells. In amethod for performing the application, the techniques described in thefollowing reports, for example, can be utilized. Concretely, CD4+T cellsrestricted to HLA-DR are isolated, the cells specifically reacting withras peptide resulting from substitution of 12th amino acid glycine withanother amino acid in the human T cells (Jung, S. J. Exp. Med. 173, 273,1991), and a CTL (cytotoxic T lymphocyte) recognizing a peptideconsisting of eight amino acids including the mutation site for 61thamino acid mutation can be derived from a mouse immunized with arecombinant vaccinia virus capable of producing ras protein, which hasmutation at 61th amino acid (Skipper, J., J. Exp. Med. 177, 1493, 1993).In addition, in a mouse immunized with a soluble mutant ras proteinprepared by gene recombination, the in vivo proliferation of tumor cellshaving the same mutation is suppressed (Fenton, R. G., J. Natl. CancerInst. 85, 1294, 1993), and a CTL showing cytotoxic activity againsttumor cells expressing the same mutant ras can be obtained fromsplenocytes sensitized with the mutant ras peptide (Peace, D. J., J.Exp. Med. 179, 473, 1994). On the other hand, the bcr-abl chimericprotein, which is often detected in chronic myelocytic leukemia,possesses high tyrosine kinase activity and plays a key role in theonset of leukemia and the proliferation of leukemic cells. By immunizingwith a peptide in the vicinity of the fusion site of this fusionprotein, T cells reactive to this fusion protein can be obtained (Chen,W., Proc. Natl. Acad. Sci. USA 89, 1468, 1992). Moreover, antisense DNAor RNA corresponding to the fusion gene is capable of suppressing theproliferation of tumors expressing this gene in vivo (Skorski, T., Proc.Natl. Acad. Sci. USA 91, 4504, 1994).

It is therefore possible to obtain T cells reactive to a receptorprotein kinase comprising the peptide of the present invention, whereinthe peptide is encoded by a nucleic acid having tandem duplicationoccurring in the nucleotide sequence of a juxtamembrane, and to regulatethe proliferation of cells that express the above kinase by immunizingwith the above peptide.

Also, when the presence of the tandem duplication of the presentinvention is involved in cell proliferation egulation, it is possible toregulate the signaling mechanism with antisense DNA or RNA for the abovegene to regulate cell proliferation

When binding a ligand to an extracellular region, the receptor proteinkinase undergoes a conformational change to form a dimer, resulting inincreased kinase domain activity in the intracellular region, wherebyself-phosphorylation or phosphorylation of a substrate of the abovekinase takes place. In these steps, various signaling molecules areinvolved, and the information transmitted into cells causes variousbiological phenomena, such as cell morphological change, cell movement,morphogenesis, cell proliferation, malignant alteration,differentiation, and apoptosis. Acute myelocytic leukemic cells of highmalignancy have been reported to possess strong affinity to the FLT3ligand and promote cell proliferation (Piacibello, W., Blood 86, 4105,1995; Lisovsky, M., Blood 36, 22a, 1995; McKenna, H., J. Exp. Hematol.24, 378, 1996; Dehmel, U., Leukemia 10, 261, 1996). In cells expressingthe FLT3 tandem duplication mutant of the present invention, it isexpected that the system for signaling from the FLT3 ligand is highlyactivated. Hematopoietic stem cells that express the mutant aretherefore provided as a source of hematopoietic stem cells possessingstrong proliferation potential. By comparing the hematopoietic stemcells with cells expressing the normal FLT3, materials and methodssuitable for screening for various drugs can be provided.

As described above, by utilizing a method of the present invention, itis applicable to the examination and treatment of blood cell diseases,hematopoietic stem cell diseases, and other diseases.

The present invention will be hereinafter described in more detail bymeans of examples, but the present invention is not limited by thoseexamples.

Example 1 1) Analysis of FLT3 Gene Expression Pattern

On 80 cases of acute leukemia patients (50 cases of child ALL, 30 casesof adult AML), analysis of FLT3 gene expression was carried out byRT-PCR method. The primers used were designed to have nucleotidesequences as shown by SEQ ID NOs: 26 and 27 in Sequence Listing, and tocompletely cover and amplify a transmembrane region through ajuxtamembrane. By using the above primer pair, the resulting amplifiedDNA product is 366 by in length, when normal FLT3 has been transcribed.

A total RNA was extracted from a peripheral blood or myelocyte derivedfrom the above patient with a Trizol reagent (manufactured by LifeTech),followed by DNA amplification reaction using an RT-PCR kit (manufacturedby Takara Shuzo Co., Ltd.) and Thermal Cycler (manufactured by TakaraShuzo Co., Ltd.) under following conditions. cDNA was synthesized from atotal. RNA using a reverse transcriptase. In 50 μl of a reaction mixturecontaining 1 μl of the cDNA (equivalent to 40 ng of a total RNA), 200 μMdNTP mixture, 1×PCR buffer, 2 U of Taq DNA polymerase, and 20 pmol eachof the above-described primers, the above reaction mixture was heated at94° C. for 5 minutes, and thereafter repeated 35 times of a thermalcycle consisting of 64° C. for 30 seconds, 72° C. for 45 seconds, and94° C. for 30 seconds, and then finally treated at 72° C. for 5 minutes.To check quality of RNA, RT-PCR was carried out in the same mannerexcept that a pair of the primers shown by SEQ ID NOs: 28 and 29 inSequence Listing were used, with β-actin as the target. The amplifiedDNA products thus obtained were subjected to electrophoresis on 2 to 3%agarose gel (manufactured by FMC) containing ethidium bromide, anddetected under UV irradiation. One example of electrophoresis pattern isshown in FIG. 1, and the results are shown in Table 1.

TABLE 1 Number of positive Length FAB Number of mRNA expression ofmutation subtype cases examined FLT3 (%) (%) AML total 30 22 (73%) 5(17%) M1 3 2 (67%) 0 M2 9 7 (78%) 1 (14%) M3 8 5 (63%) 0 M4 5 4 (80%) 2(40%) M5 4 3 (75%) 2 (50%) M6 1 1 (100%) 0 ALL total 50 39 (78%) 0 cALL27 24 (89%) 0 pre-B ALL 13 11 (85%) 0 B-ALL 1 0 0 T-ALL 9 4 (44%) 0

It is found from Table 1 that the transcription product of the FLT3 genewas found in 39 cases (78%) of the 50 ALL cases and 22 cases (73%) ofthe 30 AML cases. Among them, the amplified DNA product longer than theexpected 366 by was detected in 5 cases (23%) of the 22 FLT3-positiveAML cases, so that a length mutation in FLT3 gene was observed.Incidentally, in four cases (M34, M155, M810, and M839), the expected366 by band and a longer band than the expected were detected. In onecase (M162), the 366 by band was not detected, and a longer band alonewas detected.

2) Analysis of Nucleotide Sequence of Length Mutation Product of FLT3Gene

To examine in more detail length mutations in the gene in the above 5cases, the amplified DNA product was purified from agarose gel, andnucleotide sequences of the exon 11 and exon 12 regions were determined.As a result, it was confirmed that these length mutations resulted fromtandem duplications in nucleotide sequences of the respectivejuxtamembrane. Concretely, a 39 by or 60 by tandem duplication withinexon 11 was found in cases M34, M162, and M839; and a 26 by tandemduplication including a 4 by (GGCA) insert was found in case M810. Inaddition, case M155 was found to have a 63 by tandem duplicationcomprising the first 16 by of axon 12 immediately after axon 11, onecytosine residue insert, and the last 46 by of axon 11. The nucleotidesequences obtained are shown in SEQ ID NOs: 6 to 10 in Sequence Listing,and a schematic view of these tandem duplications is shown in FIG. 2.

Characteristically, these tandem duplications occur in-frame, and thesemutations are reflected in the actually expressed polypeptides. Theamino acid sequences encoded by these nucleotide sequences are shown inSEQ ID NOs: 1 to 5.

3) Analysis of Nucleotide Sequence of Genomic DNA

Amplified DNA products obtained by PCR with FLT3 genomic DNA derivedfrom myelocytes from the above 5 cases of patients as templates wereanalyzed. PCR-reaction was carried out under amplification conditionssuch that 2 U of Taq DNA polymerase (Takara Shuzo Co., Ltd.) was addedto a PCR buffer containing 50 ng of genomic DNA, 200 μM of a dNTPmixture, and 20 pmol of each of primers, to make up a total volume of 50μl. For exons 10 to exon 19, each exon was individually subjected toamplification DNA reaction. The length mutation was observed in samemanner as that when mRNA was analyzed in the case where exon 11 and exon12 were amplified with primers of SEQ ID NOs: 30 and 31, and primers ofSEQ ID NOs: 32 and 33 in Sequence Listing as pairs. When the PCRproducts were purified using QIAEXII (QIAGEN), cloned into pCRII vector(Invitrogen), and subjected to nucleotide sequence analysis, similarresults to those with the nucleotide sequences of cDNA (SEQ ID NOs: 21to 25) were obtained. The results are collectively shown in FIG. 2.

Example 2 Analysis of Mutations in Juxtamembrane of Receptor ProteinKinase and their Pathologic Relationship

To analyze mutations in a juxtamembrane of the receptor protein kinaseand their pathologic relationship, the relationship between thepathologic classification of symptoms and FLT3 gene is shown in Table 1.Five cases showing tandem duplication in the nucleotide sequence of ajuxtamembrane belonged to M2 (myeloblastic, with maturation potential),M4 (myelomonocytic), or M5 (monocytic) based on the FAB classification,and all of them were cases in which patients relapse into symptom todeath even with transient symptom remission.

The nucleotide sequences of axon regions encoding tyrosine kinase domainwere also analyzed, and no mutations were found in these regions.

Therefore, it was suggested that the in-frame tandem duplications ingene region encoding a juxtamembrane of FLT3 were associated with AMLwith monocyte growth was suggested.

Also, since such length mutations were not detected in DNA samples frommyelocytes collected from three cases (M34, M162, and M810) at the timeof complete remission, the tandem duplication of the present inventionwas found to be a somatic mutation.

INDUSTRIAL APPLICABILITY

According to the present invention, there can be provided a novelreceptor protein kinase having tandem duplication mutation in thenucleotide sequence of a juxtamembrane, and its nucleotide sequence andamino acid sequence information. In addition, there can be providedpathological diagnoses, a method for examination of leukemia etc.utilizing the present invention, a kit and a reagent for examinationrelated thereto. Furthermore, there can be provided a method forregulating and analyzing conditions of proliferation anddifferentiation, malignant alteration, immune response, and signallingfor cells represented by hematopoietic stem cells and leukemia cellsutilizing the present invention, and a kit and a reagent relatedthereto.

1. A primer set composition for amplifying at least a portion of aregion of nucleic acid that encodes a polypeptide having FLT3 kinaseactivity, which comprises: at least two isolated nucleic acid oligomersthat, in a nucleic acid amplification reaction, will prime theamplification of at least one nucleic acid fragment located within thenucleic acid fragment located within the nucleic acid region encodingFLT3 kinase that is defined by SEQ ID NO: 26 and SEQ ID NO:
 33. 2. Theprimer set composition of claim 1 wherein the nucleic acid fragment islocated within the nucleic acid region encoding FLT3 kinase that isdefined by SEQ ID NO: 26 and SEQ ID NO:
 27. 3. The primer setcomposition of claim 1 wherein the nucleic acid fragment is locatedwithin the nucleic acid region encoding FLT3 kinase that is defined bySEQ ID NO: 30 and SEQ ID NO:
 31. 4. The primer set composition of claim1 wherein the nucleic acid fragment is located within the nucleic acidregion encoding FLT3 kinase that is defined by SEQ ID NO: 32 and SEQ IDNO:
 33. 5. A nucleic acid fragment composition for detectingmyelodysplastic syndrome (MDS) or leukemia in a subject, whichcomprises: at least one isolated nucleic acid fragment derived from thenucleic acid region encoding FLT3 kinase that is defined by SEQ ID NO:26 and SEQ ID NO: 33 and which comprises at least one mutation comparedto the wild-type nucleic acid sequence encoding FLT3 kinase.
 6. Thenucleic acid fragment composition of claim 5 wherein the nucleic acidfragment is derived from the nucleic acid region encoding FLT3 kinasethat is defined by SEQ ID NO: 26 and SEQ ID NO:
 27. 7. The nucleic acidfragment composition of claim 5 wherein the nucleic acid fragment isderived from the nucleic acid region encoding FLT3 kinase that isdefined by SEQ ID NO: 30 and SEQ ID NO:
 31. 8. The nucleic acid fragmentcomposition of claim 5 wherein the nucleic acid fragment is derived fromthe nucleic acid region encoding FLT3 kinase that is defined by SEQ IDNO: 32 and SEQ ID NO: 33.